T lymphocytes recognize specific antigens through interaction of the T cell receptor (TCR) with short peptides presented by major histocompatibility complex (MHC) class I or II molecules. For initial activation and clonal expansion, nave T cells are dependent on professional antigen-presenting cells (APCs) that provide additional co-stimulatory signals. TCR activation in the absence of co-stimulation can result in unresponsiveness and clonal anergy. To bypass immunization, different approaches for the derivation of cytotoxic effector cells with grafted recognition specificity have been developed. Chimeric antigen receptors (CARs) have been constructed that consist of binding domains derived from natural ligands or antibodies specific for cell-surface antigens, genetically fused to effector molecules such as the TCR alpha and beta chains, or components of the TCR-associated CD3 complex. Upon antigen binding, such chimeric antigen receptors link to endogenous signaling pathways in the effector cell and generate activating signals similar to those initiated by the TCR complex. Since the first reports on chimeric antigen receptors, this concept has steadily been refined and the molecular design of chimeric receptors has been optimized (for a review see Uherek et al., 2001). Aided by advances in recombinant antibody technology, chimeric antigen receptors targeted to a wide variety of antigens on the surface of cancer cells and of cells infected by human immunodeficiency virus (HIV) have been generated (for a review see Uherek et al., 2001).
The expression of CARs with specificity for tumor-associated or viral cell surface antigens in lymphocytes such as T cells or natural killer (NK) cells generates antigen-specific effector cells for the use in adoptive, target-cell specific immunotherapy. Such CARs are composed of a cell recognition domain such as a scFv antibody fragment for recognition of a tumor-cell surface antigen fused via a flexible linker region to an intracellular signaling domain such as CD3 zeta-chain. CAR expression retargets the cytotoxic activity of lymphocytes to tumor cells that are otherwise resistant to cytolysis by immune effector cells (Uherek et al., 2001; Uherek et al., 2002; Müller et al., 2008; Tavri et al., 2009). Thereby, gene transfer using viral vectors or physical transfection methods is of limited efficiency, resulting in only a fraction of the cells permanently incorporating and expressing the transferred gene construct. Hence, it is desirable to include a selectable marker gene in such vector constructs to allow selection and enrichment of gene-modified cells prior to therapeutic applications such as adoptive therapy.
Depending on the cell type used, the relatively low transduction efficiency of viral vectors employed for genetic modification of lymphocytes (in particular NK cells) with effector genes of therapeutic value (such as genes encoding CAR) limits the relative proportion of gene-modified cells in the transduced cell pool. In principle, inclusion of a selectable marker gene in the vector constructs would allow selection and enrichment of gene-modified cells prior to potential therapeutic applications in adoptive immunotherapy. However, available selection markers such as bacterial resistance genes and bacterial enzymes cannot be used due to their non-human origin and their potential immunogenicity. Furthermore, selection using such markers requires antibiotics or toxic reagents which must be added to the culture medium.
Therefore, the present invention aims to provide means and methods for the transfer of effector genes of therapeutic interest into mammalian (human) cells, in particular lymphocytes, utilizing a selectable marker gene of human origin which allows selective enrichment of gene-modified cells in standard culture medium without addition of toxic compounds.
Furthermore, the present invention aims to provide means and methods for medical application(s) of the mammalian (human) cells obtained thereby.